Method of diagnosis of prostate cancer

ABSTRACT

The present invention relates to structures involved in the secretory processes of reproductive tissues, including the prostatic secretory processes, and their protein products which may have used as tools for diagnosing reproductive pathology including prostate disease. The present invention also relates to reagents, such as antibodies, other ligands and oligonucleotides, for detecting these structures o their contents and to methods of diagnosing prostate pathology, including prostate cancer and prostatitis. The invention further relates to an improved tissue and cell fixation process for the detection of secretory structures in reproductive tissue. The fixation process is useful for the diagnosis of prostate, testes and renal cancer.

FIELD OF THE INVENTION

The present invention relates to structures involved in the secretoryprocesses of reproductive tissues, including the prostatic secretoryprocesses, and their protein products which may be used as tools fordiagnosing reproductive pathology including prostate disease. Thepresent invention also relates to reagents, such as antibodies, otherligands and oligonucleotides, for detecting these structures or theircontents and to methods of diagnosing prostate pathology, includingprostate cancer and prostatitis. The invention further relates to animproved tissue and cell fixation process for the detection of secretorystructures in reproductive tissue. The fixation process is useful forthe diagnosis of prostate, testes and renal cancer.

BACKGROUND OF THE INVENTION

The secretory (luminal) cells of normal prostatic glands are separatedfrom the basement membrane by a layer of inconspicuous basal cells (1).This surface layer is characterised by its tall columnar cells withbasally orientated nuclei, whose abundant apical cytoplasm synthesises abroad range of secretory proteases including prostate specific antigen(PSA) and prostatic acid phosphatase (PAP). Characteristically, thecytoplasm of surface secreting cells is optically clear to faintlyeosinophilic which distinguishes it dramatically from the amphiphilic(dark) cytoplasmic staining of most dysplastic or malignant prostaticepithelial cells (2,3). Optimal tissue staining for diagnostic purposesyields maximal cytoplasmic clarity in benign prostatic secretory cells(2), the abrupt contrast between the cytoplasmic density of cancer andthe pallor of the adjacent normal epithelium in well stained sections isoften the most striking histologic feature which delineates theboundaries of a carcinoma focus. Conversely, a frequent problem inneedle core diagnosis is the “clear cell” glandular atypical focus, inwhich confirmation of carcinoma is more difficult due to the absence ofdark cytoplasm.

The significance of cytoplasmic density in adenocarcinoma is notunderstood. In the Gleason grading system (4) retention of clearcytoplasm implies a high level of differentiation since it is arequisite feature of all Grade 1 and Grade 2 carcinomas. Furthermore,cytoplasmic clarity is also characteristic of most adenocarcinomas whicharise from the transition zone in association with nodular hyperplasia(5,6). Although dark cytoplasm is well described in dysplasia (PIN) andGleason grade 3 carcinoma, it is not specific for malignant loss ofdifferentiation since it is common in the cells of benign postinflammatory atrophy (7).

The organelles responsible for the appearance of the normal prostaticsecretory cell cytoplasm have been described as myriad tiny vesicleswhich nearly fill the cytoplasm and appear completely devoid of content(1,8). These vesicles or granules are only faintly and variablyrecognisable by routine light microscopy, depending on the stainingintensity of the faintly eosinophilic narrow septa which separate them.

Prostatic corpora amylacea (CA) are extracellular intraluminalstructures seen in most adult prostate (9). The protein source of the CAis poorly understood although within these structures a group ofsulphur-rich proteins have been previously detected (10). Similarsulphated proteins have also been detected in crystals associated withwell-differentiated carcinoma, so-called prostatic crystalloids (11).Further, amyloid possibly related to β-2 microglobulin has beenrecognized in CA (12) but, despite these basic observations the originsof these extracellular prostatic structures is yet to be determined.

Round proteinaceous deposits have also been identified in prostaticluminae (9). These deposits (3-8 μm in diameter) are rare (8 cases of166 specimens) and when noted are in close association with the luminalsurface of the benign secretory cells confined to the central region ofprostate in routinely fixed and processed tissues. The nature of thisprotein deposit is unknown but was noted to be negative for numerousprotein fractions including, prostate specific antigen (PSA), prostaticacid phosphatase (PAP), light chain immunoglobulin (κ and λ),α₁-antitrypsin and α-fetoprotein (9). Stains for mucin and silver stainswere also negative but unexpectedly positive for phloxine tartrazine.

Beyond these initial observations, the detailed structure underlying the“clear cell” cytoplasmic appearance of prostatic epithelium as well asthe relationships between this appearance and the process of prostaticexocrine secretion have never been systematically studied. Further,common and apparently fundamental alterations of structure and secretoryfunction which must underly the abrupt transition to the dark cytoplasmof most cancers are unknown.

The mammalian oviduct provides an environment that supports the gametes,the process of fertilisation, early embryonic development and thedelivery of a viable embryo to the uterus. The lumen of the mammalianoviduct is formed by a complex interdigitating system of longitudinalmucosal folds. These longitudinal mucosal folds are lined by a simplecolumnar epithelium and the morphological and biochemicalcharacteristics of this epithelium are controlled by ovarian steroids.At the time of ovulation this lining epithelium consists of fullydifferentiated columnar ciliated and secretory cells. Approximately40-50% of the epithelial cells are secretory. Secretory glands areobserved in the apical regions of the cells. Several secretory productshave been identified which enhance sperm motility, viability, binding tozona pellucida and enhance the rate of zona pellucida penetration.(Verhage et al 1997 Characteristics of an oviductal glycoprotein and itspotential role in fertility control. J. Reproduction and FertilitySupplement 51:217-226).

SUMMARY OF THE INVENTION

The present inventors have now developed improved techniques forvisualising prostatic cytoplasmic structure and the mechanism of cellsecretion. These improved techniques have led to the surprising findingthat secretory granules which are found in the luminal cells of normalprostate glands are absent in prostate carcinomas. These findingsindicate that the prostate secretory granules may provide an importanttool in the diagnosis of prostate cancers.

The present inventors have also identified a link between PSG,decapitated cytoplasmic body (DCB), eosinophilic bodies (EB) and corporaamylacea (CA) structures. Briefly, the normal secretory cell cytoplasmis filled with brightly eosinophilic PSG measuring about 1 μm indiameter and densely concentrated in the apical third of the cell. Thisapical compartment represents an apocrine secretory bleb. Periodicdetachment of blebs carries packages of secretory granules (PSG) intothe lumen where the receptacles fragment liberating their contents. Theluminal cytoplasmic compartment (bleb), emptied of its protein enzymes,becomes a decapitated cytoplasmic body (DCB), a partly collapsed,faintly basophilic membrane with remnant cytoplasm. The DCB shrinks toform a sphere with a thickened, brightly eosinophilic surface casing,the eosinophilic body (EB). This structure may dissolve in luminalsecretions, but it is also observed that it may adsorb to the surface ofthe corpus amylaceum (CA).

Unexpectedly, the present inventors have found that in prostaticadenocarcinomas, the entire secretory apparatus is substantially absent;neither PSG, DCB nor EB are found. An inability to form corpora amylaceaarises from this fact. Prostate carcinomas may therefore becharacterised by a significant reduction in levels, or absence, of anyone of the structures associated with prostate secretions.

The present inventors have found that PSGs, EBs, DCBs and CAs may bereadily visualised in normal prostate tissue which has been fixed instrong glutaraldehyde, or a substance which produces similar cytoplasmicfixation to that produced by strong glutaraldehyde.

Accordingly, in a first aspect the present invention provides a methodof processing a tissue sample for analysis, the method comprisingexposing the tissue sample to a composition which produces substantiallyidentical cytoplasmic fixation to that produced by glutaraldehyde at aconcentration of at least 2.0% and/or which provides substantiallyidentical preservation of secretory granules as that provided byglutaraldehyde at a concentration of 2.0%.

In a preferred embodiment of the first aspect, the tissue sample isreproductive or renal tissue, more preferably reproductive tissue.Preferably, the reproductive tissue is prostatic, testes, fallopian tubeor oviduct tissue.

In a further preferred embodiment, the histological analysis comprisesanalysing the tissue for the presence of secretory granules.

In a second aspect the present invention provides a method of diagnosingprostate pathology in a subject which method comprises

(i) fixing a sample of prostate tissue from the subject in a fixativewhich produces substantially identical cytoplasmic fixation to thatproduced by glutaraldehyde at a concentration of at least 2.5%; and

(ii) analysing the sample for the presence of PSG and/or EB and/or DCBand/or CA structures, or the contents of any one or more of thesestructures.

It will be appreciated that a reduced number of PSG and/or EB and/or DCBand/or CA structures, or the contents thereof, in the sample isindicative of prostate disease in the subject.

In a preferred embodiment, the method of the second aspect is used todiagnose prostatitis or prostate cancer in the subject.

In one embodiment of the second aspect, the analysis in step (ii) isperformed by light microscopy. In this embodiment, the tissue ispreferably stained with a stain such as haematoxylin and/or eosin.

In a further embodiment of the second aspect, the analysis in step (ii)involves electron microscopy.

In yet a further embodiment of the second aspect of the presentinvention, the analysis in step (ii) involves immunostaining. Theimmunostaining may involve immuno detection of PSA or PAP.Alternatively, the immunostaining may involve immuno detection of PSGand/or EB and/or DCB and/or CA structures.

In a further preferred embodiment of the first and second aspects, thefixative produces substantially identical cytoplasmic fixation to thatproduced by glutaraldehyde at a concentration of between 2.5% and 6%,more preferably at a concentration of between 3% and 5%. The fixativemay comprise glutaraldehyde at a concentration of between 2.5% and 6%,more preferably at a concentration of between 3% and 5%.

In a further preferred embodiment of the first and second aspects, thefixative composition comprises an aqueous solution of glutaraldehyde ata concentration of between 2.5% and 6%, a metallic salt and a bufferstabiliser, the composition having a pH of between 5.7 and 5.75.

In a further preferred embodiment of the first and second aspects, thefixative further comprises phenol, preferably in a concentration rangesof from about 2.0 to about 3.0 g/l, more preferably around 2.5 g/l.

In a further preferred embodiment of the first and second aspects, themetallic salt is selected from the group consisting of zinc sulphate,copper sulphate, barium sulphate, cobalt chloride, barium chloride,potassium chloride, mercuric chloride and lead chloride. Preferably, themetallic salt is zinc sulphate.

In a further preferred embodiment of the first and second aspects, theconcentration of the metallic salt ranges from 3.0 to 20.0 g/l, morepreferably around 13.0 g/l.

In a further preferred embodiment of the first and second aspects, thebuffer comprises one or more acetic acid compounds. Preferably, thebuffer stabilizer comprises sodium acetate at a concentration of about0.2M and acetic acid at a concentration of about 0.2M.

In a further preferred embodiment of the first and second aspects, thefixative further comprises one or more components selected from thegroup consisting of:

Detergents such as SDS, Tween (0.001%-1.0%),

Azone (laurocaprame 1-dodecylazacyclo-hepton-2-one) 3% w/v or1-geranylazacyclohepton-2-one 3% w/v,

Liposomes,

Sodium taurocholate 40-0.25 μM; (which may include:

Cholesterol 0.2 mM-0.075mM

Oleicacid 1 mM-0.25mM

Synthetic phospholipids, eg phosphocholin 14-18 10-30 mM as mixedmicells),

Solution C24 (polyoxyethene-24-cholesterol-ether),

Polyethylene glycol 200 dilaurate (0.1-10%),

Menthol 1% w/v,

Mercaptoethanol (0.0025%),

Glycerol trioleate,

Terpene penetration enhancers (for example 1,8-cineole, methane,(+)-limonene, nerolidol),

Medium chain fatty acids (caproate C6, C8 caprylate, C10 caprate, C12laurate),

Trichloroactic acid (0.5-5.0%),

Metallic salts (for example, zinc sulphate, potassium chloride, calciumchloride, zinc chloride) (3-30%),

Dimethylsulfoxide (0.1-20%),

Mono and disaccharides (glucose),

Urea, and

Methyl salicylate.

In a third aspect the present invention provides a histological fixativecomposition comprising an aqueous solution of glutaraldehyde, a metallicsalt and a buffer stabiliser, the composition having a pH of between 5.7and 5.75.

In a preferred embodiment of the third aspect, the amount ofglutaraldehyde ranges from about 2.5% and about 6%, more preferablybetween about 3.5% and about 5%, by volume of the composition.

In a further preferred embodiment of the third aspect, the fixativecomprises phenol, preferably in a concentration ranges of from about 2.0to about 3.0 g/l, more preferably around 2.5 g/l.

In a further preferred embodiment of the third aspect, the metallic saltis selected from the group consisting of zinc sulphate, copper sulphate,barium sulphate, cobalt chloride, barium chloride, potassium chloride,mercuric chloride and lead chloride. Preferably, the metallic salt iszinc sulphate.

In a further preferred embodiment of the third aspect, the concentrationof the metallic salt ranges from 3.0 to 20.0 g/l, more preferably around13.0 g/l.

In a further preferred embodiment of the third aspect, the buffercomprises one or more acetic acid compounds. Preferably, the bufferstabilizer comprises sodium acetate at a concentration of about 0.2M andacetic acid at a concentration of about 0.2M.

In a further preferred embodiment of the third aspect, the fixativecomposition further comprises one or more components selected from thegroup consisting of:

Detergents such as SDS, Tween (0.0.01%-1.0%),

Azone (laurocaprame 1-dodecylazacyclo-hepton-2-one) 3% w/v or1-geranylazacyclohepton-2-one 3% w/v,

Liposomes,

Sodium taurocholate 40-0.25 μM; (which may include:

Cholesterol 0.2 mM-0.075mM

Oleicacid 1 mM-0.25mM

Synthetic phospholipids, eg phosphocholin 14-18 10-30 mM as mixedmicelles),

Solution C24 (polyoxyethene-24-cholesterol-ether),

Polyethylene glycol 200 dilaurate (0.1-10%),

Menthol 1% w/v,

Mercaptoethanol (0.0025%),

Glycerol trioleate,

Terpene penetration enhancers (for example 1,8-cineole, methane,(+)-limonene, nerolidol),

Medium chain fatty acids (caproate C6, C8 caprylate, C10 caprate, C12laurate),

Trichloroactic acid (0.5-5.0%),

Metallic salts (for example, zinc sulphate, potassium chloride, cobaltchloride, calcium chloride, zinc chloride) (1-30%),

Dimethylsulfoxide (0.1-20%),

Mono and disaccharides (glucose),

Urea, and

Methyl salicylate.

In a particularly preferred embodiment the fixative is prepared asfollows:

(i) Phenol (2.5 g) is dissolved in 50 ml of distilled water. The phenolsolution is added to 200 ml of glutaraldehyde (25%). The pH of thesolution is adjusted to 5.8 by the dropwise addition of 5M NaOH.

(ii) Zinc sulphate (15 g) is dissolved in 250 ml of distilled water. Thezinc sulphate solution is then admixed with 470 ml of 0.2 M sodiumacetate and 30 ml of 0.2 M acetic acid. The solution is adjusted to a pHof 5.6-5.75.

(iii) The solutions from steps (i) and (ii) are admixed and ifnecessary, the pH and is adjusted by the addition of NaOH to about 5.7.

Preferably, the final solution is filtered before use.

In a fourth aspect the present invention provides an isolated prostatesecretory granule (PSG).

By “prostate secretory granule” or “PSG” we mean a vesicle which isproduced in and secreted from normal prostatic secretory cells.

In a preferred embodiment the PSG has a diameter of 800-1200 nm. In afurther preferred embodiment, the PSG has a granular electron dense corewithout internal membranes.

In a further preferred embodiment, the PSG is eosinophilic.

In a further preferred embodiment, the PSG is glycoprotein and sulphurrich.

In a fifth aspect the present invention provides an isolated prostatecell decapitated cytoplasmic body (DCB).

In a further preferred embodiment, the DCB is glycoprotein and sulphurrich.

In a sixth aspect the present invention provides an isolated prostateeosinophilic body (EB).

In a preferred embodiment, the EB is glycoprotein and sulphur rich.

In a further preferred embodiment, the EB has a diameter of between 4and 15 μm.

In a seventh aspect the present invention provides an isolated prostatecorpora amylacea (CA).

In a preferred embodiment, the CA is glycoprotein and sulphur rich.

The present inventors have found that the contents of the PSG and/or DCBand/or EB and/or CA include sulphur-rich prostatic crystalloids,extracellular acid mucin and sulphate-associated glycosaminoglycans. Thesulphate-associated glycosaminoglycans may be rich in glucosamine andgalactose. The present inventors have also characterised thesulphated-associated compounds and have identified the substance keratansulphate as the major sulphur group of PSG, DCB, EB and CA structures.

Accordingly, in an eighth aspect the present invention provides anisolated keratan sulphate-associated compound derived from a PSG and/orDCB and/or EB and/or CA.

One particular keratan sulphate-associated compound has been purifiedand characterised by molecular weight analysis.

Accordingly, in a ninth aspect the present invention provides a keratansulphate-associated compound derived from a PSG, the compound having amolecular weight of about 70-75 kDa.

It will be appreciated by those skilled in the art that a PSG and/or DCBand/or EB and/or CA structures or the contents of these structures maybe used to generate binding ligands, such as antibodies against a PSGand/or DCB and/or EB and/or CA or the contents thereof. These bindingligands may be used in turn as diagnostic tools in the differentiationof normal and malignant prostate tissue, in situ or by their presence inbodily fluids.

Accordingly, in a tenth aspect the present invention provides a bindingligand, preferably an antibody, directed against a PSG and/or DCB and/orEB and/or CA or the contents thereof.

The term “antibody” is to be construed as covering any specific bindingsubstance having a binding domain with the required specificity for thesecretory structure. Thus, the term covers antibody fragments,derivatives, functional equivalents and homologues of antibodies,including any polypeptide including an immunoglobulin binding domain,whether natural or synthetic. Chimaeric molecules including animmunoglobulin binding domain, or equivalent, fused to anotherpolypeptide are therefore included.

It is also possible that the PSG and/or EB and/or DCB and/or CAstructures include nucleic acid molecules. In this case, as would bereadily understood by those skilled in the art, the presence of thesestructures or their contents in tissue could be detected usingoligonucleotide probes. The oligonucleotides could also be used in theamplification of the nucleic acid contents, for example, by PCR. Thepresent invention also provides such oligonucleotides.

In an eleventh aspect the present invention provides a method ofanalysing tissue for pathology, the method comprising detecting thepresence of secretory granules in a sample of the tissue.

In a preferred embodiment of the eleventh aspect, the tissue sample isreproductive tissue. Preferably, the reproductive tissue is prostatic,testes, fallopian tube or oviduct tissue. More preferably, the tissue isprostatic tissue.

In a further preferred embodiment of the eleventh aspect the pathologyis prostate cancer or prostatitis.

In a further preferred embodiment of the eleventh aspect the analysisinvolves fixing the tissue sample with a composition according to thethird aspect.

In a twelfth aspect the present invention provides a method ofdiagnosing prostate pathology in a subject which method comprisesanalysing a fluid sample from the subject for the contents of PSG and/orEB and/or DCB and/or CA structures, or the contents thereof. It will beappreciated that a decrease in any one of the structures of the contentsthereof (when compared to a fluid sample from a subject without prostatedisease) in the fluid sample is indicative of prostate disease. The bodyfluid may be blood, serum, semen or urine.

In a preferred embodiment of the twelfth aspect, the disease is prostatecancer or prostatitis.

In a further preferred embodiment of the twelfth aspect the analysisinvolves immunoanalysis using a binding ligand, preferably an antibody,according to the tenth aspect of the present invention.

In a further preferred embodiment of the twelfth aspect, the contents ofany one or more of the structures comprises a sulphate-associatedcompound according to the eighth or ninth aspects.

In a thirteenth aspect, the present invention provides a method ofmonitoring the effectiveness of the use an anticancer agent in thetreatment of prostate cancer in a subject which method comprisesobtaining sequential samples of fluid from the subject over a period oftime of treatment and detecting the levels of PSG and/or EB and/or DCBand/or CA structures, or the contents thereof, in the sequentialsamples.

In a preferred embodiment of the thirteenth aspect of the presentinvention, the fluid sample is derived from blood serum, seminal fluid,or urine.

It is known that a number of anticancer agents act by inhibiting theassembly of microtubules in tumour cells (13). It now also appears thatinhibition of microtubule assembly disturbs secretion of granules, suchas prolactin granules, from secretory glands (14). Accordingly,anticancer agents may inhibit the secretion of PSGs from prostate tumourcells.

Accordingly, in a fourteenth aspect the present invention provides amethod of screening an agent for anticancer activity, which methodcomprises

(i) exposing a sample of prostate tumour cells to the agent, and

(ii) monitoring the cells over time for the presence of PSG and/or EBand/or DCB and/or CA structures, wherein the presence of one or more ofthe structures in the cells indicates that the agent has anticanceractivity or allows maturation of cancer cells, thereby making themsusceptible to other agents.

In a preferred embodiment, the prostate tumour cells are cultured cells.In a further preferred embodiment, the cultured cells are obtained bytransformation of normal prostate luminal cells.

In a fifteenth aspect the present invention provides a method ofscreening an agent for anticancer activity, which method comprises

(i) exposing a sample of prostate cells to a transforming substance,wherein the level of exposure is sufficient to transform the prostatecells into prostate tumour cells,

(ii) exposing the cells to the agent and

(ii) monitoring the cells over a period of time for the presence of PSGand/or EB and/or DCB and/or CA structures, wherein the maintenance ofone or more of the structures in the cells over the period of time, oran increase in the level of one or more of the structures over theperiod of time, indicates that the agent has anticancer activity.

The prostate cells may be exposed to the agent simultaneously with thetransforming substance, or subsequent to exposure to the transformingsubstance.

The transforming substance may be any substance which transforms normalcells to tumour cells. Examples of suitable transforming substancescomprise the Epstein Barr Virus.

In a further preferred embodiment, the cells are monitored for a periodof 7 days, more preferably 28 days.

Throughout this specification, the word “comprise”, or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 X-ray diffraction analysis of sub-cellular and extra-cellularprostatic fractions.

FIG. 2 Effect of keratan sulphate on a mixed lymphocyte reaction.

DETAILED DESCRIPTION OF THE INVENTION

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will now be described with referenceto the following Examples.

EXAMPLE 1 Identification of Prostate Sectretory Granules (PSGs)Materials and Methods

Twenty four radical prostatectomy specimens were received fresh eitherin the Auckland Hospital laboratory [19 cases] or at Stanford Universitylaboratory [5 cases]. The prostates were orientated vertically, a probeinserted through the urethra and a transverse section cut through thelobe thought to harbour the main tumour mass. Hyperplastic tissue fromthe transition zone (TZ) as well as tumour and benign tissue from theperiphery was sampled. In three cases, two with coexistent peripheralzone (PZ) carcinomas, well-circumscribed bright yellow TZ tumours wererecognised and were sampled separately. Tissue samples from each areawere fixed in the following solutions; 95% ethyl alcohol, 4% and 10%buffered formaldehyde, 1%, 3%, and 5% buffered glutaraldehyde, 6%mercuric chloride in 1% formaldehyde (B5), and picric and acetic acid in10% formaldehyde (Bouin's solution). After 12-18 hours all fixed tissueswere processed through graded alcohols to paraffin wax. In furtherdescriptions buffered glutaraldehyde in concentrations greater than 2.5%is referred to as “strong glutaraldehyde”.

Three micron sections were cut and stained with routine haematoxylin andeosin (H&E), anti-prostate specific antigen (PSA; Dako Corp, Denmark,1:100), anti-prostatic acid phosphatase (PAP; Dako Corp, Denmark, 1:100)and cytokeratin AE1/AE3 (Dako Corp, Denmark, 1:100). In the three casesof TZ cancer, samples from the tumours and adjacent benign prostatictissues were snap frozen in cold hexane (−78° C.) and 4 micron sectionswere stained for lipids using Sudan IV and Sudan black B stains.

For electron microscopy, one mm tissue fragments from tumour and benigntissues of six cases including the three TZ cancers, were fixed ineither 3% buffered glutaraldehyde or 4% buffered formaldehyde. Tissueswere post fixed in 1% osmium tetroxide, dehydrated through gradedalcohols and embedded in epoxy resin. Ultra-thin sections (90 nm) werecut, then stained with uranyl acetate and lead citrate and viewed in atransmission electron microscopy at 100 kv.

Ultrastructural immunostaining for PSA was performed on benign andmalignant tissues from two of the six cases examined by electronmicroscopy. Samples were fixed in strong glutaraldehyde for 12 hours andfollowing sucrose impregnation, frozen thin and semi-thin sections werecut on a vibrating ultramicrotome. Antigen recognition was demonstratedwith strepavidin/gold (Zymed Inc. 1:10).

PSGs may be isolated from fresh prostate tissue which is teased througha stainless steel mesh to separate the epithelial cells and theircontents from the stroma. Cells and cytoplasmic fragments can then becollected in a buffered sucrose solution which is then subjected toultracentrifugation. This method will provide a pure isolate ofgranules. Similarly, DCPs, EBs and CAs may be isolated at lower speedcentrifugation. Electron microscopy will confirm correct isolationspeeds.

Results

Light Microscopy

In all cases, benign prostatic secretory cells fixed in ethyl alcohol,4% and 10% formaldehyde, 1% glutaraldehyde, mercuric chloride andBouin's solution showed abundant clear or palely eosinophilic cytoplasm.In contrast, benign surface cells fixed in strong glutaraldehydesolutions contained brightly eosinophilic cytoplasmic prostaticsecretory granules (PSG) which, although sometimes concentrated towardsthe luminal surface of the cell frequently filled the entire cellcytoplasm. The contrast between eosinophilic granule staining andbackground was greatly intensified by thorough washing of the H&Esections in running tap water for 45 minutes which removed all traces ofeosin bar that staining the granules. In the strong glutaraldehyde fixedtissue, many secretory cells showed apical cytoplasmic buds and otherswere associated with cytoplasmic decapitations, both structures filledwith eosinophilic PSG. Although identifiable in alcohol, formaldehyde ormercuric chloride fixed tissue these projections were less easilyvisualised due to their marked pallor and inconsistent membranestaining.

In areas of high grade dysplasia PSG were markedly reduced in number(90%+) or absent. Also no granules were seen in 17 of 22 peripheral zoneGleason grade 3 and 4 invasive cancer samples. Four of the remainingfive samples showed only isolated clusters of a few PSG in the apicalregions of individual cells. There remained a single Grade 3 cancersample in which the cells contained frequent cytoplasmic PSG; about halfthe density seen in benign cells. An adjacent formaldehyde fixed tissuesample in this isolated case showed it to have clear cell morphology.

The remaining PZ cancers and all dysplasias after either formaldehyde orglutaraldehyde fixation showed uniformly dark (amphiphilic) cytoplasm,but in contrast to formaldehyde, strong glutaraldehyde fixed benignglands adjacent to tumour cells contained red PSG.

Clear cell TZ carcinoma showed no granular cytoplasmic material. Onecase of this total series an area classified as atypical adenomatoushyperplasia (adenosis) was recognised and despite an infiltratingarchitectural pattern, cytoplasmic granules were evident in reducednumbers (30-40% of those seen in benign glands) throughout the cellcytoplasm, suggesting a diagnosis of benign atypia.

Fat stains on frozen sections confirmed that the cytoplasm of most cellsin clear cell TZ carcinoma were filled with numerous supranuclear lipidvacuoles. In contrast, benign cells contained almost no lipid except foran occasional lipid droplet consistently located below the nucleus andadjacent to the basal cell layer. In clear cell cancers processedroutinely after formaldehyde fixation, abundant supranuclear fatdroplets produced an appearance almost identical to that of benign“clear” cells. In contrast after strong glutaraldehyde fixation, thepersistent clear cytoplasm of the cancer provided histologic delineationof the tumour focus by its boundary of benign red granular cells.

Immunostains

After strong glutaraldehyde fixation, cytoplasmic granules in benignepithelium showed sharply delineated immunoreactivity for both PSA andPAP with approximately 50% of all granules staining intensely for thesemarkers. The immunoreactivity within granules was discrete and intensein contrast to negative or weakly stained background cytoplasm. Somesecretions within the lumen also retained a finely granularimmunoreactivity. Counterstaining with eosin confirmed that all apicalgranules were immunostained while basal and mid zone granules wereusually negative. In contrast the cytoplasm of the benign secretorycells fixed in formaldehyde, alcohol, mercuric chloride and Bouin'sstained diffusely for these prostate markers without any evidence ofgranularity.

With few exceptions, carcinoma cells fixed in strong glutaraldehydeshowed no granular staining for either PSA or PAP.

Electron Microscopy The prostatic secretory granules (PSG) identified bylight microscopy in the tissue fixed in strong glutaraldehyde weremembrane bound organelles 900-1000 nm in diameter and contained aelectron dense granular material. No internal membrane structures wereobserved. These granules were identified basally in small numbersadjacent to the nucleus in close proximity to Golgi and roughendoplasmic reticulum and nearly filled the cytoplasm more apically.

In many cells the apical cytoplasm was partly separated from the rest ofthe cell by inward extension of the apical cell membrane; thesecytoplasmic luminal projections containing many granules often mergedwith the luminal prostatic secretions. This was seen even in cells wherethe cytoplasmic bud had not yet separated from the underlying cell.Electron microscopic examination of formaldehyde fixed normal secretorycells tissue showed fragmented and collapsed membrane bound structuresin an identical distribution to those granules seen in strongglutaraldehyde fixed tissue.

In strong glutaraldehyde fixed tissue, immunogold deposition indicatedlocalisation of PSA and PAP specifically to many of the apical granulesof benign secretory cells without marking the intervening cytoplasm.Cancer cells fixed in strong glutaraldehyde did not contain anygranules, instead an increased concentration of ribosomes andendoplasmic reticulum was seen throughout the cytoplasm. Immunogoldstaining recognised PSA in the cytoplasm particularly near the cellmembrane.

Discussion

The distinctive cytoplasmic clarity of normal prostatic secretoryepithelium is revealed by our data to be the manifestation of a fixationartefact. Formaldehyde and a number of alternate routine fixativesdamage the epithelial cells, disrupting their apparently fragilesecretory granules that lose their contents. We are able to prevent thisdamage by prompt fixation in strong glutaraldehyde, which preserves theintact secretory granules (PSG) and reveals their brightly eosinophiliccontents. Distinctively, the granules retained full intensity of stainafter tap water washing for a period far longer than needed to removeall other traces of eosin from the slide. This unusual selectivestaining persistence is also known to characterise other acidphosphatase secreting protease granules such as those of the eosinophilleucocyte (15,16). Hence, we propose that both cationic proteins andstructural phospholipids of the secretory granules should be furtherinvestigated as likely determinants of this unusual behaviour.

Ultrastructurally, prostatic secretory granules (PSG) were about 1000 nmin diameter, a dimension consistent with their visualisation by lightnicroscopy. They contained a granular electron dense core withoutinternal membranes or structure. With both light and electron microscopyPSA and PAP were localised exclusively to the contents of intactsecretory granules. This observation depends upon fixation in strongglutaraldehyde and therefore this report represents the first definitivelocalisation of these important exocrine products.

The PSG which we describe were the only secretory organelles identifiedin this Example. In a previous study very occasional, still largereosinophilic masses were identified in the cytoplasm of some largetransition zone ducts (9). These ductal structures were negative for PSAand PAP and are presumed to be degenerate PSG or accumulations of theirstructural components; they were not seen in any tissue samples fromthis current series.

Although prostatic secretory granules have not been previously describedin detail, ultrastructural observations of seminal plasma have reportedvariably sized membrane bound secretory structures called “prostasomes”measuring 20-250 nm in diameter and identified ultrastructurally inseminal plasma (17,18) and often partitioned into clusters. They werebelieved to be secreted from the cell by a process of exocytosis ordiacytosis. By contrast, the PSG identified in this paper were 1000 nmin diameter, showed no internal subdivision, and displayed an apocrinesecretion without evidence of exocytosis or diacytosis. There does notappear to be a direct relationship between the intracytoplasmicstructures seen in this study visible by light microscopy and thoseminute prostasomes previously identified ultrastructurally in theseminal plasma.

Some features of normal prostatic granule formation and maturation werealso visualised in our sections. Basally near the Golgi apparatus,granules were sparse and stained with eosin alone (negative for PSA andPAP). With increasing distance from the cell base, proteaseimmunostaining became more intense along with increasing PSG density.This is consistent with the initial production of a pre-PSA molecule,which acquires biochemically activity and antigenicity with proximity tothe cell apex.

The secretory process for the PSG has been described in broad outlineand was confirmed here in detail. The area where PSG were most closelyspaced and intensely stained was the apical third of the secretory cellcytoplasm. This apocrine secretory compartment was furtherimmunohistochemically delineated by an abrupt absence of cytokeratinstaining, indicating a loss of apical cytoplasmic filaments. Theresultant fragile apical sac was observed to regularly disintegratein-situ at the cell surface without detaching and its contents mergedwith the luminal secretion as individual PSG. These apical details werelost with formaldehyde fixation, but even with routine preparations theragged, uneven and focally indistinct luminal cell border of theepithelium identified the distinctive features of the normal prostaticcell secretory process.

By contrast, in dysplasia (PIN) and in moderately well differentiatedcarcinomas with dark (amphiphilic) cytoplasm (Gleason 3), there weredramatic deviations from the normal secretory cell structure. Except forsparsely scattered PSG in a few cells, dysplasia and cancer alike showedno evidence of granule synthesis. Immunostainable PSA and PAP werevariably demonstrated in cancer, but were released free into thecytoplasm and tended to be more sharply localised along the surface cellmembrane. Further investigation of this drastic alteration in cellfunction within adenocarcinoma cells might help to explain phenomenasuch as epitope differences in cancer-produced PSA (19) as well as thegreater affinity of PSA in cancer for alpha-1-antichymotrypsin (20).

In clear cell carcinomas of the transition zone, retention of normal PSGproduction was expected from observations on formaldehyde fixed tissue.But surprisingly granule production suffered the same fate ofobliteration seen in dark cell carcinomas and dysplasias. Cytoplasmicclarity here depended on an alternate and aberrant pathway ofdifferentiation in which the secretory product was lipid contained intiny secretory droplets. On this basis transition zone clear cellcarcinomas can now be definitively classified as a biologically distinctentity by their commitment to functions which have no parallel in thenormal prostate nor in carcinomas with dark cytoplasm.

Clear cell areas of peripheral zone cancers in this series showed thesame cytoplasmic features as those in the transition zone. There was asingle exception (4% of cases) in which a peripheral zone clear cellcarcinoma area continued to produce abundant normal PSG. In all otherclear cell cancers, the depletion of PSG and concurrent establishment ofa lipid synthesis pathway would suggest the culmination of a complexsequence of epigenetic changes whose early stages might well predate theemergence of invasive carcinoma. Nothing is yet known about possiblepremalignant alterations in clear cell carcinoma since special fixationis needed to reveal this pathway.

If strong glutaraldehyde is used in routine fixation, clear cellcarcinomas will persist as foci with clear cytoplasm sharply outlined bya border of benign glands with intensely red granular cytoplasm. Acommercial fixative Ultrim II (American Histology Reagent Company LodiCalif.) may used rather than glutaraldehyde because it produces the samecytoplasmic fixation, is less toxic and preserves finer nuclear detail.This method of fixation will provide a practical manner in futurestudies to more reliably distinguish clear cell malignancies, toevaluate premalignant phases of clear cell cancer and to bettercharacterise atypical glandular proliferations now classified asadenosis or atypical glandular hyperplasia (21,22) whose biologicalstatus remains obscure at this time. Further, this method can enhancethe evaluation of prostatic tissue on a gland by gland basis in smallsamples such as needle biopsies.

EXAMPLE 2 Identification of Eosinophilic Protein Deposit (EPD) Materialsand Methods

Hyperplastic prostatic tissue (4 cases) as well as tumour and benigntissue from the periphery of the gland (2 cases) were collected asdescribed in Example 1. Tissues from each area were divided into half;one half then fixed in 3% buffered glutaraldehyde, the other in 4%buffered formaldehyde. After 12-18 hours, fixed tissues were dividedinto 4 mm cubes and then dehydrated through a process of freeze-drying;the fixed tissue was immersed in liquid butane (−200° C.) thendehydrated under vacuum conditions for 48 hours. Finally, the specimenswere gradually warmed to +58° C. in the vacuum and immersed in liquidwax. Control tissue processed routinely from adjacent areas wasavailable in each case. The micron sections were cut and stained withroutine H&E, alcian blue (pH 2.5), Congo red, phloxine tarterazine,anti-prostate specific antigen (PSA; Dako Corp, Denmark, 1:100) andanti-prostatic acid phosphatase (PAP; Dako Corp, Denmark, 1:100) and β-2microglobulin Dako Corp, Denmark, 1:100). In an attempt to identifychemical differences between the different eosinophilic structures, testand control samples were subjected to X-ray diffraction studies. Threemicron sections were cut on onto melinex film, electrostatically coatedwith carbon film. Thereafter, the sections were examined with ablack-scatter detected in a scanning electron microscope (PSEM 500) at25 KV and a spot size of 0.25 microns. Specific protein deposits i.e. CAand crystalloids as well as PSG were targeted and analyzed using an EDAXdetection unit (P500 EDS) for 100 seconds.

Results

In all benign tissue the prostatic luminae were filled with roundintensely eosinophilic proteinaceous deposits (EPD) measuring 4-15 μ indiameter. These were most numerous in lumen of larger ducts and wereoften seen surrounding and merging with prostatic corpora amylacea.These structures were in contrast to PSG were negative on immunostainingfor PSA and PAP. Stains for alcian blue confirmed weak staining of PSGbut intense staining of EPD. The alcian blue staining of the EPD wasconcentrated in a beaded corona surrounding a pale protein core. Alltumours and control formaldehyde fixed benign tissue showed noassociated PSG or intraluminal EPD.

X-ray diffraction studies showed abundant sulphur-rich material in bothPSG and EPD (FIG. 1) in freeze dried material fixed in glutaraldehyde.

Discussion

PSG is a complex protein constituent of benign secretory cells which isnow recognized to be intimately associated with a previouslyuncharacterized alcohol-soluble eosinophilic protein deposit (EPD). Thefailure to previously recognize EPD (9) is based on their alcoholsolubility. Analyses performed in prostate tissue protected from alcoholelution (current freeze drying techniques) confirms that most glandscontain abundant EPD. The deposits of EPD fill the ducts of the glandand in turn surround and merge with intraluminal corpora amylacea.

EPD consists of a protein core, which is intensely eosinophilic and likePSG, is resistant to tap water washing. Its outer rim is composed of aglycoprotein rich, alcian blue positive material which also stains moreintensely with phloxine tartrazine. Phloxine tartrazine identifies viralinclusions although keratin and granules of intestinal paneth cells arealso strongly stained.

Prostatic crystalloids, a common feature of well-differentiatedcarcinoma, share some of the staining characteristics of EPD in thatthey are intensely eosinophilic and resistant to water elusion. Incontrast to EPD they lack a glycoprotein component, the absence of whichmay relate to an alternate secretory mechanism of glycoproteins bycancer cells and the subsequent presence of abundant extracellularacidic mucin.

EXAMPLE 3 Prostate Secretory Apparatus: PSG, DCP, EB and CA StructuresMaterials and Methods

Prostatic tissue with nodular hyperplasia (5 cases), together withbenign and carcinoma tissue from the peripheral zone of the prostate (6cases) were collected from eight radical prostatectomy specimensreceived in this laboratory. Tissues from each area were divided intothree portions; one fixed in 3% buffered glutaraldehyde (“Solufix”®),the second in 4% buffered formaldehyde and the third kept unfixed onice. After 12-18 hours, fixed tissues were divided into 4 mm cubes andwere processed in three separate ways; (i) routinely to wax paraffinthrough standard alcohol dehydration, (ii) through propylene oxide toepoxy resin and (iii) to paraffin wax by dehydration through a processof freeze-drying. This latter method relied on the fixed tissue beingimmersed in liquid propane (−120° C.), dehydrated under vacuumconditions for 18 hours and then warmed to +58° C. and immersed inliquid wax. In addition to material taken from fresh radicalprostatectomy specimens, archival material from prior samples taken atradical prostatectomy were also examined. These samples consisted ofthree peripheral zone cancers as well as three transitional zonecarcinomas rich in prostatic crystalloids. Samples from these six caseswere bisected at the time of surgery and half was fixed in 3%glutaraldehyde the other half in 4% formaldehyde. All six cases wereprocessed routinely through alcohol and embedded in wax.

The unfixed prostate samples (benign and malignant) were homogenized andcentrifuged through a 0.25M sucrose solution. Pellets and supernatantwere collected after centrifugation at 15,000 g, 18,000 g and 20,000 g.All pellets were divided for electron microscopy and routinepolyacrylamide gel electrophoresis. Further, prostatic concretions wereextracted from benign tissue and purified through multiple washings insaline solution. Light microscopy was used to confirm successfulextraction and polyacrylamide gel electrophoresis was performed. Samplesof PSG and concretions were submitted to high performance exchangechromatography (Dionex DX 500 carbohydrate system) to assesscarbohydrate and amino acid content. Washed concretions were alsosubjected to X-ray analysis using a Rigaku RU-300 rotating anode X-raygenerator producing Cu K_(∝) radiation of wavelength 0.154 nm withfocusing optics. Diffraction patters, which are dependent on thepresence of true crystals, were recorded for 20 minutes by a“Mar-Research 345” image plate area detector.

Routinely processed glutaraldehyde and formalin fixed tissues as well asfreeze dried and plastic embedded material were cut (plastic embeddedtissue at 1 μm and paraffin embedded tissue at 3 μm) and stained withroutine haematoxylin and eosin (H&E) as well as histochemical andimmunostains. These included periodic acid Schiffs (PAS), alcian blue(pH 2.5, pH 1.0), Congo red, phioxine tartrazine, anti-prostate specificantigen (PSA; Dako Corp, Denmark, 1:100), anti-prostatic acidphosphatase (PAP; Dako Corp, Denmark, 1:100), β-2 microglobulin (DakoCorp, Denmark, 1:100), and sialosyl-Tn antigen (STn; Dako 1:50). Heatantigen retrieval was used for β-2 microglobulin as recommended in theproduct brochure.

X-ray diffraction was performed with the aid of scanning electronmicroscopy; two selected formalin fixed samples, three glutaraldehydefixed samples as well as two freeze dried glutaraldehyde-fixed tissuesamples were assessed targeting the PSG, concretions, crystalloids andother eosinophilic structures. Three-micron sections were cut ontomelinex film and electrostatically coated with carbon. Thereafter, thesections were examined with a back-scatter detector in a scanningelectron microscope (PSEM 500) at 25 kV and a spot size of 0.25 μm.Results were analyzed using an EDAX detection unit (P500 EDS) for 100seconds. Control areas in the gland lumen, cytoplasm (between PSG) andsurrounding stroma were also targeted.

Results

Histology

In all benign tissues fixed in glutaraldehyde, numerous bright red PSGwere visualised in the surface secretory cells and showed concentrationin the apical portion of the cell cytoplasm. These apical apocrinecompartments (blebs) of different cells were in various stages offormation and shedding, giving an uneven surface border. After detachingfrom the luminal surface of the secretory cell, the evolving blebschanged from tear drop shape to a spherical contour and after losingtheir eosinophilic contents (PSG) they became pale blue membrane ghostswhich filled the gland lumens and which we called “decapitatedcytoplasmic bodies” (DCB). The DCB were 8-15 μm in diameter andcentrally they merged with the prostatic secretions and becameindistinct (Table 1). None of these observations could be made onformalin-fixed tissue which had empty appearing cell cytoplasm and glandlumens.

In tissues processed by freeze drying and stained routinely withhaematoxylin and eosin, many DCB acquired a discrete brightlyeosinophilic rim, contrasting with a central darker red core. As aresult many of these DCB could be distinguished from central prostaticsecretions and we termed these eosinophilic bodies (EB). Centrally, theywere most numerous often overlapping and ranging in size from 4-8 μm. Inlumens of larger ducts EB could be seen fusing with the surface ofexisting corpora amylacea, adding in a lamellar fashion to the corporadiameter, or forming small intra-luminal concretions. None of the abovefindings could be visualized in formalin-fixed tissue.

Among prostatic adenocarcinomas, Gleason grade 3 with amphiphiliccytoplasm and architecturally simple gland structures comprises the mostcommon histological acinar pattern. After glutaraldehyde fixation andeither plastic embedding or freeze drying, the cytoplasm in nearly allsuch malignant cells was almost completely devoid of the PSG which fillbenign cell cytoplasm. PSA and PAP were accordingly displaced from theirnormal location and were diffuse in the cytoplasm with strongaccentuation at the apical plasma membrane. Luminal DCB and EB werealmost never seen (Table 1).

Clear cell carcinoma is the commonest histologic pattern for transitionzone carcinomas, but, in the present series, it appeared mainly inperipheral zone as islands of cancer with clear cytoplasm and Gleasongrade of 3 or less. The line of transition to surrounding amphiphilicgrade 3 cancer was usually sharp. Near this border on the amphiphilicside, the dark tumour cells were often accompanied by intraluminalmucin; within the clear cell cancer nidus, mucin was almost alwaysabsent, and occasional groups of lumens contained dense, brightlyeosinophilic crystalloids. Many of these cancer lumens also contained agranular pale eosinophilic protein matrix that surrounded thecrystalloids (12) and was most easily recognized in glutaraldehyde fixedtissues. The cytoplasm of most clear cells had few or no PSG, but glandlumens containing crystalloids were surrounded by epithelium having ahigher concentration of PSG but never approaching that of benignepithelium.

TABLE 1 Frequency of Prostate Associated Intra- and ExtracellularProtein Structures in benign, dysplastic and neoplastic tissues. BenignDysplasia Carcinoma Prostate Secretory +++ ± ± Granules (PSG)Decapitated +++ ± − Cytoplasmic Bodies (DCB) Eosinophilic Bodies ++ − −(EB) Corpora Amylacea (CA) + − − Crystalloids − − ± Frequency: 75-100%(+++), 50-75% (++), 10-50% (+), 1-10% (±) and 0% (−)

Analysis of Luminal Contents vs Cell Cytoplasm

PSG contained 20% sugar by weight. This comprised 1.76 nmol/μl ofglucosamine, and 0.64 nmol/μl of galactose and 0.4 nmol of galactosamine(total of 2.8 nmol/μl). Proteins were estimated at 0.1 nmol/μl and thusthe molar ratio of monosaccharides/amino acids was 30:1. Amino acidanalysis did no show significant concentrations of sulphur containingfractions (<3%) and the most common amino acids identified were glycine(8.9%), proline (8.9%), leucine (8.8%) alanine (7.9%), and valine(7.9%). Equimolar concentration of sulphate to glucosamine wasidentified confirming the components of sulphated glycosaminoglycans ofproteoglycans.

Analysis of the corpora amylacea also confirmed a large concentration ofglucosamine (1.2 nM/μl) and galactose (0.86 nM/μl) in relation toprotein (1.0 nM/μl). Amino acid analysis confirmed significantconcentrations of alanine, valine and proline and as in the PSGsulphur-containing amino acids represented a minority of the total aminoacids (<3%). An equimolar concentration of sulphate (1.1 nM/μl) toglucosamine was also detected. Monosaccharide/amino acid ratio ofcorpora amylacea was 2:1.

X-ray crystallography of the corpora amylacea demonstrated widedispersion patterns consistent with a true biological crystal composedof very small sub-units (less than 200 Da). This pattern isrepresentative of alternating disaccharide bases as may be contained inglycoprotein chains and is too small to represent most common cellproteins.

Abundant sulphur-rich material was identified by electron microscopydirected X-ray diffraction. In alcohol dehydrated or freeze-dried tissueafter glutaraldehyde fixation, PSG, DCB and EB contained highconcentrations of sulphur despite low concentrations of sulphurcontaining amino acids. Areas of cytoplasm between PSG and areas ofstroma in freeze-dried tissues were targeted and failed to reveal anysulphur and were therefore used as control background graphs.Importantly, the presence of sulphur in DCB that had lost eosinophilicstaining in routine processing indicated that eosinophilia and sulphurmay depend upon the presence of two separate molecules; the first asulphated glycosaminoglycan the second a cationic protein. Further,sulphur was present at comparably high levels in corpora amylacea and inluminal crystalloids of clear cell carcinomas.

PSG, EB and corpora amylacea were all moderately to strongly PASpositive regardless of tissue preparation. In glutaraldehyde-fixed,freeze-dried preparations, alcian blue (pH=1) weakly stained PSG andstrongly stained EB, with staining limited to the brightly eosinophilicrim. Corpora amylacea were alcian blue positive (pH=1 & 2.5). Clear cellcarcinomas were consistently negative for mucin with alcian blue(pH=2.5) or STN antibody, a mucin tag seen in most prostate cancers(23). Their granular luminal contents were weakly alcian blue positive(pH=1), as were scattered foci within crystalloids. By contrast, therewas intense mucin positivity (STn and Alcian Blue, pH=2.5) in carcinomaswith amphiphilic cytoplasm. It consistently filled cancer gland lumensand STn was focally present within malignant cell cytoplasm.

TABLE 2 Histochemical, immunostaining and X-Ray analysis of prostateassociated granules (PSG) and extracellular protein deposits ProstateDecapitated Eosino- Prostatic Secretory Cytoplasmic philic CorporaCrystal- Granules Bodies Bodies Amylacea loids Eosin +++ − +++ ++ +++PAS + − ++ ++ + Alcian Blue ± −  ++¹ ++  +² (pH 1.0) Congo Red +++ − +++++ +++ Phloxine ++ − +++ +++ +++ Tartrazine β2 ± − − ± − micro- globulinSTn Antigen − − − − − PSA/PAP  +++³ − − − − Sulphur +++ +++ +++ ++ +++¹Outer Rim staining ²Amorphous material surrounding crystalloids ³ApicalPSG ± − Initial focal weak staining, eliminated with avidin-biotinblocking. + moderate staining. ++ strong staining. +++ intense staining

PSG, DCB, EB, corpora amylacea and crystalloids all stained stronglywith Congo Red and with phloxine tartrazine (Table 2). However, therewas no apple green birefringence under polarized light with any of thesestructures.

Immunostains for PSA, PAP and Keratan Sulphate exclusively stained thePSG of benign glands while in amphiphilic carcinoma it followed ingeneral, the same luminal distribution of cancer associated mucin. Weakimmunostaining for β-2 microglobulin was identified in the PSG andcorpora amylacea. However, on repeated stains, this apparentimmunoreactivity to β-2 microglobulin was significantly reduced oreliminated with the use of avidin-biotin blocking (Dako Corp, DenmarkX0590). Immunoreactivity for mucin using anti-STn was negative in allbenign epithelial tissues. Keratan sulphate stains identified the PSG,eosinophilic rim of the DCB and CA. This confirmed the identity of thePSG associated GAG protein as Keratan sulphate. Cancer cells wereusually weakly stained or failed to stain with Keratan Sulphate.

PSG extracted through a sucrose gradient were confirmed by electronmicroscopy to be highly concentrated in the 18,000 g extraction.Standard polyacrylamide gel electrophoresis of 18,000 g extractions ofbenign tissue and cancer tissue identified numerous bands in each group.Western blot preparation using PSA confirmed immunolocalisation to a33-37 kDa band in all extracts but repeated attempts using β-2microglobulin failed to recognize any protein fraction. Western botanalysis for Keratan Sulphate confirmed a defined band at 75-80 KDa.Despite multiple efforts, no definite protein bands were seen in gelsrun from dissolved concretions. Instead, only stained smears were seensuggesting a lack of any sizable pure protein fractions within theprostatic concretions. This pattern however, is frequently seen in gelscontaining glycosaminoglycans.

Discussion

Since 1779 when Morgagni described the corpora amylacea as thecoagulated humour of the prostate (24) little has been known about thenature of the secretory process in prostatic epithelium and its dramaticdifferences between benign and malignant epithelium. Research in thisarea has undoubtedly been hindered by the high susceptibility of themorphologic components of the secretory apparatus to severe distortionby routine fixation. Yet in addition, there has been reluctance toconsider the exclusive presence of corpora amylacea in the lumens ofbenign glands and conversely limitation of luminal mucin or crystalloidsto cancer as possibly important clues to some of the phenotypic changesaccompanying malignant transformation. Much more has been written aboutthe very uncommon presence of crystalloids or mucin in benign glandlumens than about their possible biologic relationship to process in themalignant glands which they are almost invariably associated.

With the aid of careful fixation and tissue processing, we have beenable to derive morphologic evidence about the normal prostatic secretoryprocess and its disruption in carcinoma. The prostatic secretory granule(PSG) is a 1 μm, membrane bound structure originating from the Golgiapparatus and containing the bulk of the many different species ofprostatic secretory products including PSA, PAP and now a copious amountof GAG protein in the form of Keratan Sulphate. The PSG accumulate inthe apical third of the cell, are secreted by a unique apocrinemechanism and are finally dispersed in the gland lumen.

The luminal cytoplasmic compartment (bleb), emptied of its proteinenzymes, becomes a decapitated cytoplasmic body (DCB), a partlycollapsed, faintly basophilic membrane with remnant cytoplasm. The DCBshrinks to form a sphere with a thickened, brightly eosinophilic surfacecasing, the eosinophilic body (EB). This structure presumably maydissolve in luminal secretions, but it is also observed that it mayadsorb to the surface of the corpus amylaceum and the EB appears to beits chief mechanism for adding bulk and lamellar structure. The loss ofeosinophilia within decapitated cytoplasm therefore represents the lossof one of the PSG constituent proteins, likely to represent a cationicprotein enzyme. The eosinophilia of the EB rim by contrast is due to aglycoprotein complex. Further, EB previously recognized (9) as rarestructures within the prostate devoid of PSA and occurring only incentral areas, represented an artefact of standard fixation where theircentral location protected them from complete elution as evidenced bycurrent glutaraldehyde fixation and freeze-drying techniques.

Endogenous biotin reactivity recognized within the PSG and corpora wasgreatly enhanced by heat antigen retrieval. Recognition of this activityhas previously been confined to the kidney, brain and liver tissue (25)but not prostate gland. Its presence has implications for allimmunostains on prostate tissues when using heat retrieval as it canproduce falsely positive results. Accordingly, β-2 microglobulinpositivity was almost completely eliminated by avidin-biotin blocking.The diagnostic accuracy of the polyclonal serum used in this andprevious studies (12) is further questioned by a negative result onrepeated western blotting, a technique not previously used in theevaluation of prostatic β-2 microglobulin. The concept that corporaamylacea arise from urinary proteins (12) has been discounted by thisobservation.

The corpora amylacea were found to be composed of more carbohydrate thanprotein (monosaccharide/amino acid—2:1) and its high concentrations ofglucosamine, and galactose parallels the sugar and sulphate content ofcytoplasmic PSG confirming the histological findings that the lattercontributes to the formation of the former. The carbohydrate andsulphate content is typical of a glycosaminoglycan and from the aminoacids present in the protein backbone, is entirely consistent withKeratan sulphate (26). Glycosaminoglycans are responsible for theformation of protease granules in other well characterised biologicalsystems including leucocytes, basophils and mast cells where the anionicproteoglycan binds and temporarily stabilizes the cationic proteases(27).

In prostatic adenocarcinomas, with few exceptions and whatever the gradeor cytoplasmic type, the entire secretory apparatus was absent; neitherPSG nor DCB were found. An inability to form corpora amylacea arisesinexorably from this fact. In the usual moderately differentiatedcancers with dark cytoplasm (Gleason grade 3), luminal mucin is oftenabundant, but there is no evidence of its cytoplasmic precursor. Mucindoes not appear to be derived from distortion of benign cellcarbohydrate processing. PSA and PAP are both diffusely cytoplasmic andluminal plasma membrane concentrated. It can be speculated that theseenzymes previously linked to anionic glycosaminoglycans are intransformed cells somehow linked to mucin metabolism in a novel membranetransport process.

Fields of amphiphilic Grade 3 cancer often abut or surround clear-cellcarcinoma foci. Their cytoplasm is superficially identical to that ofnormal benign epithelium, but they are filled with lipid vacuoles ratherthan PSG¹ and hence represent an unique differentiation pathway. Thepresence of small numbers of PSG and focal Keratan Sulphate staining insome clear cells indicates that to a minor degree the normal secretorypathway is still open. Perhaps it is significant that in clear cellcarcinoma gland lumens with higher PSG concentrations in surroundingcells, eosinophilic sulphur containing crystalloids are especiallycommon. Perhaps crystalloids represent a remnant product of normalsecretion, with preservation of the cationic strongly eosinophilic PSGprotein, which has crystallized in an altered environment. The usuallyhigh sulphur content of the glycosaminoglycan of the PSG pathway hasalso been identified in crystalloids (11) and crystalloids share similarhistochemical staining patterns with PSG.

As GAGs such as Keratan Sulphate usually complex with specific proteins,the Keratan Sulphate associated protein core may represent a secretedproduct of the PSG which may be useful in both the diagnosis and therapyof prostatic conditions.

EXAMPLE 4 Prostate Secretory Granules (PSG) as a Marker of Prostatitis

Chronic non-infective prostatitis is a poorly defined disease processwhose aetiology is currently unknown. Serum from a group of four menwith histologically proven prostatitis (granulomatous) was subjected toWestern blot analysis to identify any possible antibody the PSG or itscomponents. A group of normal males and females were used as controls.Early results indicate the presence of an auto-antibody against aprotein component of PSG which has a weight of between 55 and 65 KDa.this antibody was not detected in normal controls. This may prove auseful diagnostic marker in diagnosis of chronic prostatitis.

EXAMPLE 5 Chromophobe Renal Carcinoma

Chromophobe cell renal carcinoma is a distinct tumour type with uniquemorphologic and cytogenetic features (28-32). The major histologicalfeature, which characterizes these carcinomas, is their voluminous cellcytoplasm that has a pale finely reticular quality and contrasts withwell-defined cell borders. An eosinophilic variant is also recognised inwhich tumour cells contain an additional complement of mitochondria.Distinction from conventional renal carcinoma is totally dependent onthe identification of these subtle features in routinely stainedsections and the diagnosis is only then confirmed by special stainsand/or electron microscopy. These tumours unlike conventional (clearcell) renal cell carcinomas contain abundant muco-polysaccharide thatreacts positively with Hale's colloidal iron (29), they lack neutralfat, and although irnmunoreactive for cytokeratin, they are negative forvimentin staining. Ultrastructurally the tumour is characterised bynumerous cytoplasmic micro-vesicles, thought to arise from outpouchingsof the cytoplasmic mitochondria (33,34). These tumours appear to have asignificantly more favourable outcome when compared with conventionalclear or granular cell (30,31,35).

Case Report

A 71-year-old male presented with recent onset abdominal discomfort andan incidental 4 cm mass was identified on CT scan in the upper pole ofthe left kidney. Past medical history included a previous diagnosis ofprostatic carcinoma for which the patient had received external beamradiation. At this time there was no evidence of prostatic tumourrecurrence and serum PSA was 0.1 ng/ml (normal range <4 ng/ml).

Gross Features

Following a left radical nephrectomy the fresh specimen was bisected anda uniform tan colored partly cystic tumour was identified arising fromthe superficial cortex of the upper pole. The tumour appeared partlyencapsulated and did not involve the renal vessels.

Materials and Methods

Tumour tissue was fixed in both 4% buffered formaldehyde well as“Solufix” (Tissue Technologies, Australia) a commercial tissue fixative,while frozen unfixed tissue was sectioned and stained for neutral fatusing Sudan IV. Processed tissues (both Solufix and formaldehyde fixed)were cut at 4 μm for routine haematoxylin and eosin stains, PAS, PearlsPrussian Blue, Von Kossa as well as stains for mucopolysaccharides(Hale's colloidal iron). Immunostains for Vimentin (Dako Corp, Denmark,1:100) were performed and cryostat sections were stained for lipid withSudan IV. For electron microscopy, thin sections (60-90 nm) were stainedwith uranyl acetate and lead citrate and were examined in a Phillip's410LS Transmission Electron Microscope at 80 KV. X-ray microanalysis wasalso carried out with an EDAX detection system (Moran ScientificSoftware, Australia).

DNA Isolation

To isolate DNA from tumour tissue and normal kidney tissue, 20 μmparaffin sections were cut, dewaxed in xylene and then DNA was preparedas previously described (36). Loss of heterozygosity was determined byPCR amplification of polymorphic microsatellite markers and gelelectrophoresis (37). Markers used in this study were against regionspreviously reported as being useful in the differential diagnosis ofrenal cell (38). These included D1S2883 (chromosome 1), D2S202 (2q32),D3S1675 (3p), D3S1497 (3p22-3p21.3), D3S1514 (3p21-3p14.2), D3S1447(3p21), D3S1478 (3p21.3-3p21.2), D3S1581 (3p21.2), D6S305 (6q27),D10S1239 (10q23-10q24), D13S317 (13q22), D17S559 (17p13), D17S855(17q12-17q21), D21S267 (21q22.1-21q22.3).

Results

Routine formaldehyde fixed tissue confirmed a tumour growing in solidsheets as well as focally having a tubuloalveolar pattern. Tumour cellshad clear bulky cytoplasm, moderate nuclear atypia and indistinct cellmembranes. In contrast, tumour tissue fixed in Solufix demonstrated goodpreservation of cell cytoplasm that had a fine reticular qualitycontrasting with sharply defined cell borders. Dark smudged nuclei seenin the formaldehyde fixed tissue were not identified in materialpreserved in Solufix. In contrast, nuclei were clear with distinctlyclumped chromatin. As .cell membranes were sharply focused, thevoluminous cytoplasm created a characteristic “plant cell like”appearance, a feature not easily recognised in routine formaldehydefixed sections. An unusual feature seen in all sections was theformation of numerous psammoma bodies. These seemed to arise from the“center” of tubulo-alveolar structures, which frequently containedeosinophilic material studded with small flecks of calcium.Histochemical and immunostains were similar in both Solufix and formalinfixed samples. Stains for Hale's colloidal iron was strongly positive inthe cell cytoplasm and stained the “central” amorphous deposits andemerging psammoma bodies. These structures also stained strongly withPrussian blue stain (Pearls) and Von Kossa confirming their ferrous andcalcium content. PAS stain confirmed occasional focal positivity forglycogen and immunostains for vimentin were negative in all tumourcells. Cryostat sections were negative for neutral fat (Sudan IV).

Electron Microscopy

The tumour consisted of closely apposed polygonal cells often arrangedin “tubule-like” structures. A prominent basal lamina surrounded eachcluster of cells while small stunted microvilli projected into thenarrow intercellular space as well as in lumen-like spaces. The“central” proteinacious material that was noted on light microscopy toform psammoma bodies was recognised as a primarily stromal/basementmembrane structure, which contained electron dense granular material.This material ranged in size from 25-280 nm in diameter and wasoccasionally observed as a fibrillar feather-like structure. Largergranules were also identified and found on EDAX analysis to containsignificant peaks for iron, calcium and phosphate confirming the lightmicroscopic findings. The main ultrastructural feature of the tumourcells were the presence of vacuolar and vesicular structures in thecytoplasm ranging in size from 500-1600 nm. They consisted of a closedsmooth membrane and were either round, ovoid or irregular in shape and asmall proportion contained “inner vesicles” measuring 150-300 nm indiameter. The vesicular membranes were always smooth and ribosomes werenever seen on their surface.

Molecular Biological Assessment No loss of heterozygosity (LOH) wasobserved at D3S1447. The patient was homozygous at D3S1675, D3S1497,D3S1514, and D3S1478. LOH at chromosome 3p is commonly observed inconventional (clear cell) non-papillary renal cell carcinomas. No LOHwas observed at D1S2883, D2S202, D6S305, D10S1239, D13S317, D17S559 orD21S267. The patient was homozygous at D17S855. LOH at these loci isalso commonly observed in chromophobe renal cell carcinomas andoccasionally oncocytoma.

Discussion

Chromophobe renal carcinoma has a significantly better prognosis thanconventional renal carcinoma (clear cell type) therefore making itmandatory that distinction be made in each case. Although the grossappearance is often suggestive, diagnosis rests with its characteristiclight microscopic appearance. The abundant reticular cytoplasm withdistinct cell borders often provides the clue to embark on confirmatoryhistochemical and immunostains or in selected cases, electronmicroscopy. In this case, these light microscopic features were notobvious in formaldehyde fixed tissue and were only noted after fixationin Solufix. The histochemistry, immunostains and electronnmicroscopicfeatures all strongly support the diagnosis of chromophobe carcinoma andalthough the molecular markers do not identify any of the many possiblemarkers of chromophobe carcinoma, they also do not support the diagnosisof a conventional “clear cell” carcinoma.

A feature of this tumour is the presence of numerous psammoma bodies.These are often seen in papillary renal carcinoma (39) and occasionallyin oncocytoma (7%) (30). The mechanism of psammomatous calcification iscontroversial and the accepted concept of origin from necrotic papillahas been questioned by the presence of hydroxyappatite suggestingintracytoplasmic evolution of psammoma bodies (40). In this case originappears to be within the stroma adjacent to tumour cells in whichaccumulation of mucopolysaccharides, calcium ions and haemosiderininitiates the evolution of the psmmoma body. The association of psammomabodies with haemosiderin pigment has not been previously described andmay be important factor in this unusual form of calcification.

In summary this case represents a renal tumour that has the features ofa chromophobe carcinoma, but would have been classified as conventional“clear cell” carcinoma²⁴ if fixed only in formaldehyde. After fixationin “Solufix” cytological features suggestive of chromophobe renalcarcinoma were easily appreciated. As formaldehyde is the mainstay oftissue fixation it is highly likely that many other renal tumourvariants have been designated conventional “clear cell” renal cellcarcinoma without further investigation. As the classification of renalcarcinoma is incomplete and constantly changing (41) variant tumourssuch as this case require identification not only to refine and improvethese tumour divisions but also because of their prognostic differences.Future fixation of at least small samples of tumour in Solufix may avertfuture misdiagnosis with their subsequent prognostic implications.

EXAMPLE 6 Characterisation of Keratan Sulphate-Associated ProteinMaterials and Methods

1. Benign protatic tissue was homogenised and then diluted with an equalvolume of buffered sucrose.

2. The homogenate was centrifuged at 750×g for 10 minutes at 4° C.

3. The supernatant was collected and each 3 ml was layered onto adiscontinuous sucrose gradient consisting of 3 ml each of 20%, 40% and60% (w/v) sucrose buffered with 10 mM Tris phosphate.

4. The tubes were centrifuged at 20,000 rpm for 3hr at 4° C. in aBeckman SW41 rotor.

5. Fractions were collected from above the 20% sucrose layer(supernatant) and the interaces between the 20% and 40% (interphase 1)and the 40% and 60% (interphase 2) layers.

6. Material from interphase 2 was recognised by electron microscopy tocontain intact PSGs.

Results

Interphase 2 material was run on a standard PAGE gel and western blotanalysis confirmed keratan sulphate binding to several major proteinbands. The largest of these (70-75 kD) was cut from the gel.

EXAMPLE 7 Effect of Keratan Sulphate on Immune Responses Materials andMethods

The function of keratan sulphate in prostate tissue is unknown. It isknown, however, that GAG compounds are important in the prevention ofautoimmune bonne diseases (forms of arthritis). In order to determinewhether keratan sulphate may have an immune regulatory effect it wasassessed by intorducing it into a mixed lymphocyte reaction. At aconcentration of 20 μg/ml, a reduction of 50% was observed at lymphocyteratios of 10, 30 and 90:1. At markedly diluted lymphocyte ratiosof >270:1 no effect was evident (see FIG. 2).

These results confirma significant inhibitory effect of keratan sulphateon the immune response. This may be important in normal prostatefunction, maintaining viability of sperm and for fertility.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

References

1. McNeal J E, Prostate. In: Sternberg S S, ed. Histology forpathologists, 2nd Edition, Chapter 42, Philidelphia-New York, RavenPress, 997-1017, 1997.

2. Epstein J I, Prostate biopsy interpretation, Second Edition,Philadelphia-New York, Lippincott-Raven Press, 1-12, 87-133, 1995.

3. Epstein J I. Diagnostic criteria of limited adenocarcinoma of theprostate on needle biopsy. Hum Pathol 26: 223-229, 1995.

4. Gleason D F. The Veterans Administration Co-Operative UrologicalResearch Group; histological grading and clinical staging of prostaticcarcinoma. In Tannenbaum M ed., Urological Pathology. Philadelphia: Lea& Febinger, 171-197, 1977.

5. McNeal J E. Redwine E A. Freiha F S. Stamey T A. Zonal distributionof prostatic adenocarcinoma. Correlation with histologic pattern anddirection of spread. Am J Surg Pathol 12(12): 897-906, 1988.

6. Brandes D Lesions of the prostate, In G Hill ed Uropathology, NewYork, Churchill Livingstone, 1989.

7. Cina S J, Epstein J I. Adenocarcinoma of the prostate with atrophicfeatures. Am J Surg Pathol 21(3): 289-295, 1997.

8. de Vries C R, McNeal J E, Bensch K. The prostatic epithelial cell indysplasia: an ultrastructural perspective. Prostate 21: 209-221, 1992.

9. Cohen R J, Verhaart M J S, Taylor L F. Intracytoplasmic inclusions inbenign prostatic epithelium. Arch Pathol Lab Med 118: 1030-1031, 1994.

10. Drachenberg C B. Papadimitriou J C. Prostatic corpora amylacea andcrystalloids: similarities and differences on ultrastructural andhistochemical studies. Journal of submicroscopic Cytology & Pathology.28(2):141-50, 1996 April.

11. Del Rosario A D. Bui H X. Abdulla M Ross J S. Sulfur-rich prostaticintraluminal crystalloids: a surgical pathologic and electron probex-ray microanalytic study. Human Pathology. 24(11):1159-67, 1993.

12. Cross P A. Bartley C H. McClure J. Amyloid in prostatic corporaamylacea. Journal of Clinical Pathology. 45 (10): 894-7, 1992.

13. Jiang, J. D., Wang, Y., Roboz, J., Strauchen, J., Holland, J. F. andBekesi, J. G. Inhibition of microtubule assembly in tumour cells by3-bromoacetylamino benzoyl urea, a new cancericidal compound. CancerRes. 58:2126-2133, 1998.

14. Niwa, J., Minase, T., Mori, M. And Hashi, K. Immunohistochemical,electron microscopic, and morphometric studies of human prolactinomasafter short term bromocriptine treatment. Surg. Neurol. 28(5):339-344,1987.

15. Bass D A. Lewis J C. Szejda P. Cowley L. McCall C E. Activation oflysosomal acid phosphatase of eosinophil leukocytes. Lab Investig44(5):403-409, 1981.

16. Egesten A, Calafat J, Weller P F, Knol E F, Janssen H, Walz T M,Olsson. Localization of granule protein in human eosinophil bone marrowprogenitors. Internat Arch Allergy Imunol 114: 130-138, 1997.

19. Grey E, Edgar S G, Cohen R J. Immunohistochemical detection of PSAantigen using a monoclonal antibody and polyclonal serum: a quantitativeassessment using image analysis. Brit J Urol 78:104-108 1996.

20. Wolff J M, Borchers H, Effert P J, Habib F K Jakse G. Free-to-totalprostate-specific-antiigen serum concentrations in patients withprostate cancer and benign prostatic hyperplasia. Brit J Urol 78:409-413, 1996.

21. Epstein J I. Adenosis (atypical adenomatous hyperplasia):Histopathology and relationship to carcinoma. Pathol Research & Practice191(9): 888-898, 1995.

22. Gaudin P B. Epstein J I. Adenosis of the prostate: Histologicfeatures in needle biopsy specimens. Am J Surg Pathol 19(7): 737-747,1995.

23. Kuwabara H, Uda H, Takenaka I. Immunohistochemical detection ofsialosyl-Tn antigen in carcinoma of the prostate. British Journal ofUrology 1997;80:456-459.

24. Prather G, Skinner D. Prostatic corpora amylacea. Journal of Urology1956;76:107-114.

25. Bussolati G, Gugliotta P, Volante M, Pace M, Papotti M. Retrievedendogenous biotin: a novel marker and a potential pitfall in diagnosticimmunohistochemistry. Histopathology 1997; 31(5):400-407.

26. Parmley R T Hurst R E, Takagi M, Spicer S S, Austin R L.Glycosaminoglycans in human neutrophils and leukemic myeloblasts:ultrastructural, cytochemical, immunogenic and biochemicalcharacterization. Blood 1983; 61(2): 257-266.

27. Fischer D C. Henning A. Winkler M. Rath W. Haubeck H D. Greiling H.Evidence for the presence of a large keratan sulphate proteoglycan inthe human uterine cervix. [journal Article] Biochemical Journal. 1996;320 (Pt 2):393-399.

28. Bonsib S, Lager D: Chromophobe cell carcinoma: analysis of fivecases. American Joumcal of Surgical Pathology 14:260-267, 1990.

29. Tickoo S, Amin M, Zarbo R: Colloidal iron staining in renalepithelial neoplasms, including chromophobe renal cell carcinoma:emphasis on technique and patterns of staining. American Journal ofSurgical Pathology 22:419-424, 1998.

30. Cochand-Priollet B, Molinie V, Bougaran J et al: Renal chromophobecell carcinoma and oncocytoma. A comparative morphologic,histochernical, and immunohistochemical study of 124 cases. Archives ofPathology & Laboratozy Medicine 121:1081-1086, 1997.

31. Crotty T, Farrow G, Lieber M: Chromophobe cell renal carcinoma:clinicopathological features of 50 cases. Journal of Urology154:964-967, 1995.

32. Billis A, Carvalho R, Magrini E et al: Chromophobe renal cellcarcinoma: clinicopathological study of 7 cases. UltrastructuralPathology 22:19-26, 1998.

33. Thoenes W, Storkel S, Rumpelt H-J et al: Chromophobe cell renalcarcinoma and its variants—a report on 32 cases. Journal of Pathology155:277-287, 1988.

34. Erlandson R, Reuter V: Renal tumor in a 62-year-old male.Ultrastructural pathology 12:561-567, 1988.

35. Renshaw A, Henske E, Loughlin K et al: Aggressive variants ofchromophobe renal cell carcinoma. Cancer 78:1756-1761, 1996.

36. Turbett G R, Barnett T C, Dillon E K et al: A single-tube protocolfor the extraction of DNA or RNA from paraffin-embedded tissues using astarch-based adhesive. BioTechniques 20:846-853, 1996.

37. McCulloch R, Sellner L, Papadimitriou J et al: The incidence ofmicrosatellite instability and loss of heterozygosity in fibroadenoma ofthe breast. Breast Cancer Research and Treatment 49:165-169, 1998.

38. Bugert P, Kovacs G: Molecular Differential diagnosis of renal cellcarcinomas by microsatellite analysis. American Journal of Pathology149:2081-2088, 1996.

39. Delahunt B, Eble J: Papillary renal cell carcinoma: aclinicopathologic and immunohistochemical study of 105 tumors. ModernPathology 10:537-544, 1997.

40. Murayama H, Kamio A, Imai T et al: Gastric carcinoma withpsammomatous calcification: report of a case, with reference tocalculogenesis. Cancer 49:788-796, 1982.

41. Kovacs G, Akhtar M, Beckwith B et al: The Heidelberg classificationof renal cell tunours. Journal of Pathology 183:131-133, 1997

What is claimed is:
 1. A method of diagnosing prostate pathology in asubject which method comprises (i) fixing a sample of prostate tissuefrom the subject in a fixative which produces substantially identicalcytoplasmic fixation to that produced by glutaraldehyde at aconcentration of at least 2.0%; and (ii) analysing the sample for thepresence of at least one structure selected from the group consisting ofprostate secretory granules (PSG), eosinophilic bodies (EB), decapitatedcytoplasmic bodies (DCB), corpora amylacea (CA), or the contents of anyone or more of these structures.
 2. A method as claimed in claim 1 inwhich the prostate pathology is prostatitis or prostate cancer.
 3. Amethod as claimed in claim 1 in which the protate pathology is prostatecancer.
 4. A method as claimed in claim 1 in which the analysis in step(ii) is performed by light microscopy.
 5. A method as claimed in claim 4in which the tissue is stained with haematoxylin and eosin.
 6. A methodas claimed in claim 1 in which the analysis in step (ii) compriseselectron microscopy.
 7. A method as claimed in claim 1 in which theanalysis in step (ii) comprises immunostaining.
 8. A method as claimedin claim 1 in which the fixative produces substantially identicalcytoplasmic fixation to that produced by glutaraldehyde at aconcentration of between 2.5% and 6%.
 9. A method as claimed in claim 1in which the fixative comprises glutaraldehyde at a concentration ofbetween 2.5% and 6%.
 10. A method as claimed in claim 1 in which thefixative composition comprises an aqueous solution of glutaraldelhyde ata concentration of between 2.5% and 6%, a metallic salt and a bufferstabiliser, the composition having a pH of between 5.7 and 5.75.
 11. Amethod as claimed in claim 10 in which the amount of glutaraldehyderanges from about 3.5% to about 5% by volume of the composition.
 12. Amethod as claimed in claim 10 in which the metallic salt is selectedfrom the group consisting of zinc sulphate, copper sulphate, bariumsulphate, cobalt chloride, barium chloride, potassium chloride, mercuricchloride and lead chloride.
 13. A method as claimed in claim 12 in whichthe metallic salt is zinc sulphate.
 14. A method as claimed in claim 10in which the concentration of the metallic salt raiiges from 3.0 to 20.0g/l.
 15. A method as claimed in claim 10 in which the buffer comprisesone or more acetic acid compounds.
 16. A method as claimed in claim 15in which the buffer stabilizer comprises sodium acetate at aconcentration of about 0.2M and acetic acid at a concentration of about0.2M.
 17. A method as claimed in any one of claims 10-16 in which thefixative further compnrises one or more components selected from thegroup consisting of: Detergents, Azone (laurocaprame1-dodecylazacyclo-hepton-2-one) 3% w/v or 1-geranylazacyclohepton-2-one3% w/v, Liposomes, Sodium taurocholate 40-0.25 μM Solution C24(polyoxyethene-24-cholesterol-ether), Polyethylene glycol 200 dilaurate(0.1-10%), Menthol 1% w/v, Mercaptoethanol (0.0025%), Glyceroltrioleate, Terpene penetration enhancers, Medium chain fatty acids,Trichloroactic acid (0.5-5.0%), Metallic salts, Dimethylsulfoxide(0.1-20%), Mono and disaccharides, Urea, and Methyl salicylate.